Protein drugs are the most active and fastest-growing parts of the current pharmaceutical research and development field. Recombinant protein therapeutics is different from small molecule drugs due to its structural specificity and protein molecule instability, so more strict control is needed in bio-synthesis, purification and storage steps. The instability of the protein is mainly concentrated at its C- and N-terminal, and the analysis of the variations at the C-terminus and N-terminus is particularly important.
What Is N-Terminal Pyroglutamate?
Pyroglutamate is found in many proteins, and N-terminal glutamic acid and glutamine residues can spontaneously cyclize to become pyroglutamate, or enzymatically converted by glutaminyl cyclases.
In the process of preparing antibodies, although antibodies are stable molecules, they are nevertheless subject to a variety of enzymatic and non-enzymatic degradation reactions which may occur during manufacturing and storage. What’s more, cyclization of N-terminal glutamine (Gln) to pyroglutamate (PyroGlu) has been often observed for recombinant monoclonal antibodies and leads to antibodies with lower molecular weights. So, the molecular modifications (such as N-terminal variants) for antibodies need to be detected rapidly and we can identify this modification precisely by liquid chromatography-mass spectrometry (LC-MS).
Figure 1. Formation of pyroglutamic acid from N-terminal glutamine
Analysis of N-Terminal Pyroglutamate at Creative Proteomics
At Creative Proteomics, we have developed a professional platform for analysis of N-terminal pyroglutamate. We firstly identify the N-terminal PyroGlu and determine the percentage of each variant, so as to detect the N-terminal pyroglutamate. This method is commonly accomplished by enzyme digestion followed by LC-MS to separate, identify and quantify the different forms.
Typically, to confirm the presence of N-terminal Gln or PyroGlu, antibody variants are separated by, for instance, cation exchange chromatography and collected into separate fractions, which are concentrated and analyzed by LC-MS. The molecular weights of antibody light chain, heavy chain and various proteolytic fragments which measured by LC-MS can be used to testify the presence or absence of PyroGlu by database searching. Subsequently, the identities of the N-terminal amino acid can ultimately be determined by LC-MS peptide mapping.
In addition, ESI Q-TOF mass spectrometry can directly determine the cyclized N-terminal glutamine and our tech support can help you to make it with this method.
Creative Proteomics has modified the protocols from decades of experiments, and provides robust LC-MS based analytical services. We have professional experts to help you design the best strategies and perform experiments for the N-terminal pyroglutamate analysis. Moreover, if you have any other needs, we would love to design studies to address your specific requirements. We are confident to provide high-quality data for our clients to facilitate their researches.
1. Xu, W., et al. Method to convert N-terminal glutamine to pyroglutamate for characterization of recombinant monoclonal antibodies. Anal Biochem. 2013, 436(1), 10-12.